Methods for Treating Hematopoietic Neoplasms

ABSTRACT

This invention relates to methods for treating, preventing and/or managing hematopoietic neoplasm in a subject by administering to the subject combretastatin A-4 phosphate, or a pharmaceutically acceptable salt thereof. The method may further comprise co-administering a chemotherapeutic agent.

I. CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional patentapplication No. 60/989,786, filed 21 Nov. 2007.

II. INTRODUCTION

Although chemotherapy induces remission in the majority of adultpatients with acute myeloid leukemia (AML), only small percent are curedwith conventional chemotherapy. Relapse of leukemias is in part due tothe persistence of minimal residual leukemias that remain viable withinspecialized niches, such as vascular niches. Hence, novel treatmentstrategies are urgently needed to block the interaction of hematopoieticneoplasms with activated vasculature, interfering with the establishmentof pro-leukemic niches in various organs and to eradicate resistantdisease.

Adhesion of leukemic cells to stromal cells has been shown to conferincreased resistance to chemotherapeutic agents and diminish the rate ofapoptosis of the leukemic cells. This process, named celladhesion-mediated drug resistance (CAM-DR), depends on the interactionof integrins with their ligands. Adhesion of VLA4 (very late antigen 4,α4β1) integrin-positive myeloid cells, to VCAM-1+ stromal cells is animportant mediator of CAM-DR. Indeed, expression of VLA4 by leukemiccells portends a poor prognosis and a decreased five-year survival rate.Therefore, identification of novel anti-leukemic agents that inhibitinteraction of leukemic cells with vascular cells provides novelstrategies to target organ-infiltrating, angiogenesis-dependentleukemias.

Within the marrow or in circulation, hematopoietic neoplasms are closelyassociated with endothelium, supporting establishment of neo-vessels byelaboration of angiogenic factors. In addition, leukemic cells mayactivate endothelial cells by releasing pro-inflammatory factors,including interleukin-1 (IL-1), facilitating invasion into tissues andformation of infiltrative organ disease or subcutaneous tumors, namelychloromas, thereby establishing chemotherapy-refractory leukemic minimalresidual disease.

One approach to destabilize interactions of hematopoietic neoplasms withendothelium is through disruption of the cytoskeletal organization ofthe neoplastic cells. Indeed, disruption of cytoskeletal stability ofhematopoietic neoplasms may not only promote cell death directly, butalso diminish the cellular interaction of the hematopoietic neoplasmswith vascular cells, thereby increasing sensitivity to chemotherapy.

Combretastatin-A4, a novel tubulin-destabilizing agent, was isolatedfrom the South African tree Combreturn caffrum. Combretastatin-A4 bindsto tubulin at the same site as colchicine does, but with even higheraffinity. Its pro-drug, combretastatin-A4 phosphate (CA4P) induces rapidmicrotubule depolymerization and vascular shutdown in subcutaneous solidtumors causing tumor necrosis at concentrations well below the maximumtolerated dose. CA4P also can induce apoptosis of the endothelial cellsby disengaging VE-cadherin interaction. Thus, CA4P may not only targetrapidly proliferating leukemic cells directly, but also diminishinteraction of the leukemic cells with activated endothelial cells,thereby preventing establishment of a perivascular nidus for leukemicchloromas.

The inventors demonstrate that CA4P at low, non-toxic doses,surprisingly induces rapid cell death of non-adherent leukemic cellsthrough caspase activation, mitochondria destabilization andaccumulation of reactive oxygen species (ROS), accompanied by therelease of pro-apoptotic mitochondrial membrane proteins. Additionallysingle-agent CA4P treatment is effective in eradicating bothcirculating, and vascular-adherent leukemic cells in subcutaneous andsystemic mouse models of AML, without affecting normal hematopoiesis.CA4P-treated mice had significantly prolonged survival and showed adrastic reduction of detectable leukemic cells in the marrow andperipheral circulation, and significantly decreased leukemic organinfiltration. In addition, CA4P decreases expression of VCAM-1 onendothelial cells both in vitro and in vivo, thereby decreasing leukemiccell adhesion to the vascular cells, thereby reversing drug resistance.Therefore, CA4P delivered in combination with chemotherapeutic agentsrepresents a promising novel therapeutic approach to treat hematopoieticneoplasms.

III. SUMMARY OF THE INVENTION

One aspect of the invention provides methods of treating a hematopoieticneoplasm comprising administering a therapeutically effective amount ofcombretastatin A-4 phosphate (CA4P), or a pharmaceutically acceptablesalt thereof, to a subject having a hematological malignancy.

Another aspect of the invention provides the use of combretastatin A-4phosphate, or a pharmaceutically acceptable salt thereof, for thetreatment of a hematopoietic neoplasm. The invention also contemplatesuse of combretastatin A-4 phosphate and salts thereof in the preparationof a medicament for use in treating a hematopoietic neoplasm.

Yet another aspect of the invention provides methods of treating anon-solid tumor comprising administering a therapeutically effectiveamount of combretastatin A-4 phosphate or a pharmaceutically acceptablesalt thereof, to a subject suffering from non-solid tumor.

IV. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates combretastatin A-4 phosphate (CA4P) blocking leukemiccell growth of a panel of leukemic cell lines.

FIG. 2 illustrates CA4P induction of G2/M arrest.

FIG. 3 illustrate CA4P induction of DNA fragmentation in leukemic cells.

FIG. 4 demonstrates CA4P inducing cell death without evidence ofnecrosis.

FIG. 5 demonstrates that CA4P mediated apoptosis of leukemic cells ispartially reversed by the caspase inhibitor Q-VD.

FIG. 6 illustrates the decrease in mitochondrial transmembrane potentialof leukemic cells in response to exposure to CA4P.

FIG. 7 demonstrates that Combretastatin A-4 phosphate (CA4P) inducedcell death can be partially prevented by co-incubation with ascorbicacid, an ROS scavenger, in a concentration-dependent manner.

FIG. 8 demonstrates early ROS accumulation during CA4P treatment.

FIG. 9 illustrates that CA4P mediated apoptosis of leukemic cells isreversed by inhibiting ROS and caspase pathways.

FIG. 10 provides quantification of the microvessel density in HL60 tumorsections after CA4P treatment.

FIG. 11 demonstrates that combretastatin A-4 phosphate (CA4P) improvessurvival of xenotransplanted mice with human leukemia cells.

FIG. 12 illustrates the CA4P-mediated decrease in leukemic cellcirculation in the peripheral blood and engraftment in the bone marrow,spleen, liver and lung.

FIG. 13 demonstrates that CA4P inhibits IL-1-mediated upregulation ofVCAM-1 in HUVECs.

FIGS. 14A and 14B demonstrate that CA4P reduces leukemic cell adhesionto HUVECs.

FIGS. 15A and 15B illustrate that leukemic cells adherent to HUVECs aremore resistant to CA4P.

V. DETAILED DESCRIPTION A. Definitions

As used herein, a “therapeutically effective amount” of combretastatinA-4 phosphate (CA4P), or a therapeutically acceptable salt thereof,according to the present invention is intended to mean that amount ofthe CA4P that will inhibit the growth of, or retard cancer, or killmalignant cells, and cause the regression and palliation of cancer,i.e., reduce the proliferation rate and/or the number of malignant cellswithin the body. Other desired anti-tumor effects include, withoutlimitation, the modulation of neoplasm growth rates, the enhancement ofnecrosis or hypoxia in malignant cells, reduced retention of CEPs andother pro-angiogenic cells, amelioration or minimization of the clinicalimpairment or symptoms of hematopoietic neoplasms, extending thesurvival of the subject beyond that which would otherwise be expected inthe absence of such treatment, and the prevention of neoplastic growthin an animal lacking any neoplasm formation prior to administration,i.e., prophylactic administration.

As used herein, the terms “modulate”, “modulating” or “modulation” referto changing the rate at which a particular process occurs, inhibiting aparticular process, reversing a particular process, and/or preventingthe initiation of a particular process. Accordingly, if the particularprocess is neoplastic growth or metastasis, the term “modulation”includes, without limitation, decreasing the rate at which neoplasticgrowth and/or metastasis occurs; inhibiting neoplastic growth and/ormetastasis, including tumor re-growth following treatment with ananticancer agent; reversing neoplastic growth and/or metastasis(including tumor shrinkage and/or eradication) and/or preventingneoplastic growth and/or metastasis.

“Hematopoietic neoplasm” refers to a cell proliferative disorder arisingfrom cells of the hematopoietic lineage. Generally, hematopoiesis is thephysiological process whereby undifferentiated cells or stem cellsdevelop into various cells found in the peripheral blood. In the initialphase of development, hematopoietic stem cells, typically found in thebone marrow, undergo a series of cell divisions to form multipotentprogenitor cells that commit to two main developmental pathways: thelymphoid lineage and the myeloid lineage. The committed progenitor cellsof the myeloid lineage differentiate into three major sub-branchescomprised of the erythroid, megakaryocyte, and granulocyte/monocytedevelopmental pathways. An additional pathway leads to formation ofdendritic cells, which are involved in antigen presentation. Theerythroid lineage gives rise to red blood cells while the megakaryocyticlineage gives rise to blood platelets. Committed cells of thegranulocyte/monocyte lineage split into granulocyte or monocytedevelopmental pathways, the former pathway leading to formation ofneutrophils, eosinophils, and basophils and the latter pathway givingrise to blood monocytes and macrophages.

Neoplasms of hematopoietic cells can involve cells of any phase ofhematopoiesis, including hematopoietic stem cells, multipotentprogenitor cells, oligopotent committed progenitor cells, precursorcells, and mature differentiated cells. The categories of hematopoieticneoplasms can generally follow the descriptions and diagnostic criteriaemployed by those of skill in the art (see, e.g., InternationalClassification of Disease and Related Health Problems (ICD 10), WorldHealth Organization (2003)). Hematopoietic neoplasms can also becharacterized based on the molecular features, such as cell surfacemarkers and gene expression profiles, cell phenotype exhibited by theaberrant cells, and/or chromosomal aberrations (e.g., deletions,translocations, insertions, etc.) characteristic of certainhematopoietic neoplasms, such as the Philadelphia chromosome found inchronic myelogenous leukemia. Other classifications include NationalCancer Institute Working Formulation (Cancer, 1982, 49:2112-2135) andRevised European-American Lymphoma Classification (REAL).

The term “hematopoietic neoplasm” includes, but is not limited to, acutelymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronicmyelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), hairycell leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, multiplemyeloma, and myeloplastic syndrome.

“Myeloid neoplasm” refers to proliferative disorder of cells of themyeloid lineage of hematopoiesis. Neoplasms can arise from hematopoieticstem cells, myeloid committed progenitor cells, precursor cells, andterminally differentiated cells. Myeloid neoplasms can be subdividedbased on the phenotypic attributes of the aberrant cells or thedifferentiated state from which the abnormal cells arise. Subdivisionsinclude, among others, myeloproliferative diseases,myelodysplastic/myeloproliferative diseases, myelodysplastic syndromes,acute myeloid leukemia, and acute biphenotypic leukemia.

“Lymphoid neoplasm” refers a proliferative disorder involving cells ofthe lymphoid lineage of hematopoiesis. Lymphoid neoplasms can arise fromhematopoietic stem cells as well as lymphoid committed progenitor cells,precursor cells, and terminally differentiated cells. These neoplasmscan be subdivided based on the phenotypic attributes of the aberrantcells or the differentiated state from which the abnormal cells arise.Subdivisions include, among others, B cell neoplasms, T cell neoplasms,NK cell neoplasms, and Hodgkin's lymphoma. Committed progenitor cells ofthe lymphoid lineage develop into the B cell pathway, T cell pathway, orthe non-T/B cell pathway. Similar to the myeloid lineage, an additionallymphoid pathway appears to give rise to dendritic cells involved inantigen presentation. The B cell progenitor cell develops into aprecursor B cell (pre-B), which differentiates into B cells responsiblefor producing immunoglobulins. Progenitor cells of the T cell lineagedifferentiate into precursor T cells (pre-T) that, based on theinfluence of certain cytokines, develop into cytotoxic orhelper/suppressor T cells involved in cell mediated immunity. Non-T/Bcell pathway leads to generation of natural killer (NK) cells.

The term “hematopoietic neoplasm” includes, but is not limited to, acutelymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronicmyelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), hairycell leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, multiplemyeloma, and myeloplastic syndrome.

As used herein, the term “pharmaceutically acceptable salt” includessalts that are physiologically tolerated by a subject. Such salts aretypically prepared from an inorganic and/or organic acid. Examples ofsuitable inorganic acids include, but are not limited to, hydrochloric,hydrobromic, hydroiodic, nitric, sulfuric, and phosphoric acid. Organicacids may be aliphatic, aromatic, carboxylic, and/or sulfonic acids.Suitable organic acids include, but are not limited to, formic, acetic,propionic, succinic, camphorsulfonic, citric, fumaric, gluconic, lactic,malic, mucic, tartaric, para-toluenesulfonic, glycolic, glucuronic,maleic, furoic, glutamic, benzoic, anthranilic, salicylic, phenylacetic,mandelic, pamoic, methanesulfonic, ethanesulfonic, pantothenic,benzenesulfonic (besylate), stearic, sulfanilic, alginic, galacturonic,and the like. Other pharmaceutically acceptable salts include alkalimetal cations (such as Na, K, Li), alkali earth metal salts (such as Mgor Ca), or organic amine salts (such as those disclosed in PCTInternational Application Nos. WO 02/22626 or WO 00/48606 and U.S. Pat.Nos. 6,855,702 and 6,670,344, which are incorporated herein by referencein their entireties). Particularly preferred salts include organic aminesalts such tromethamine (TRIS) and amino acid salts such as histidine.Other exemplary salts that can be synthesized using the methods of theinvention include those described in U.S. Pat. No. 7,018,987, which isincorporated by reference herein.

B. Methods of Treating Hematopoietic Neoplasms

Adhesion of leukemic cells to vascular cells may confer resistance tochemotherapeutic agents. Therefore, disruption of leukemic cellcytoskeletal stability and interference with vascular cell interactionsshould promote leukemic cell death. Indeed, as disclosed in greaterdetail below, low and non-toxic doses of combretastatin-A4 phosphate(CA4P) inhibit leukemic cell proliferation in vitro and induce mitoticarrest and cell death. Treatment of acute myeloid leukemias (AMLs) withCA4P leads to disruption of mitochondrial membrane potential, release ofpro-apoptotic mitochondrial membrane proteins (MMPs) and DNAfragmentation, resulting in cell death in part through acaspase-dependent manner. In addition, CA4P rapidly increasesintracellular reactive oxygen species (ROS), and antioxidant treatmentimparts partial protection from cell death, suggesting that ROSaccumulation contributes to CA4P-induced cytotoxicity in AML. In vivo,CA4P inhibits proliferation and circulation of leukemic cells anddiminishes the extent of peri-vascular leukemic infiltrates, therebyprolonging the survival of xenotransplanted mice, without inducinghematological toxicity. CA4P decreases the interaction of leukemic cellswith neo-vessels by down-regulating the expression of adhesion molecule,VCAM-1, thereby augmenting leukemic cell death. These data suggest that,CA4P target both circulating and vascular-adherent leukemic cellsthrough mitochondrial damage and down-regulation of VCAM-1, withoutincurring hematological toxicities. As such, combretastatin A-4phosphate provides for an effective means to treat refractoryorgan-infiltrating leukemias.

Accordingly, one aspect of the present invention provides a method oftreating a hematopoietic neoplasm, the method comprising administering atherapeutically effective amount of combretastatin A-4 phosphate, or apharmaceutically acceptable salt thereof, to a mammal suffering from ahematopoietic neoplasm. Preferably the pharmaceutically acceptable saltis a tromethamine salt of combretastatin A-4 phosphate.

Derived from the South African tree Combreturn caffrum, combretastatinswere initially identified in the 1980's as potent inhibitors of tubulinpolymerization. Combretastatin A-4 has been shown to bind a site at ornear the colchicine binding site on tubulin with high affinity. In vitrostudies clearly demonstrated that combretastatins are potent cytotoxicagents against a diverse spectrum of tumor cell types in culture.Phosphate prodrugs of combretastatin A-4 were subsequently developed tocombat problems with aqueous insolubility. Surprisingly, CA4P has alsobeen shown to cause a rapid and acute shutdown of the blood flow totumor tissue that is separate and distinct from the anti-proliferativeeffects of the agents on tumor cells themselves. A number of studieshave shown that combretastatins cause extensive shut-down of blood flowwithin the tumor microvasculature, leading to secondary tumor cell death(Dark et al., Cancer Res., 57: 1829-34, (1997); Chaplin et al.,Anticancer Res., 19: 189-96, (1999); Hill et al., Anticancer Res.,22(3):1453-8 (2002); Holwell et al., Anticancer Res., 22(2A):707-11,(2002). Blood flow to normal tissues is generally far less affected byCA4P than blood flow to tumors, although blood flow to some organs, suchas spleen, skin, skeletal muscle and brain, can be inhibited (Tozer etal., Cancer Res., 59: 1626-34 (1999)).

As used herein, the term “combretastatin A-4 phosphate” denotes acompound of the Formula I:

One implementation comprises use of a compound of Formula II,

wherein

each OR¹ and OR² independently is selected from OH, —O⁻QH⁺ and —O⁻M⁺,wherein M⁺ is a monovalent or divalent metal cation, and Q is:

a) an amino acid containing at least two nitrogen atoms where one of thenitrogen atoms, together with a proton, forms a quaternary ammoniumcation QH⁺; or

b) an organic amine containing at least one nitrogen atom which,together with a proton, forms a quaternary ammonium cation, QH⁺. Inanother implementation, wherein one of OR¹ and OR² is hydroxyl, and theother is —O⁻QH⁺ where Q is L-histidine. In another embodiment, thecombretastatin A-4 phosphate salt is a compound of Formula I, whereinone of OR¹ and OR² is hydroxyl and the other is —O⁻QH⁺ and Q istris(hydroxymethyl)amino methane (tromethamine or “TRIS”).

Another implementation comprises use of a compound of Formula II,wherein R¹ and R² are O⁻M⁺, wherein each M⁺ independently is analiphatic organic amine, an alkali metal, a transition metal, aheteroarylene, a heterocyclyl, a nucleoside, a nucleotide, an alkaloid,an amino sugar, an amino nitrile, or an nitrogenous antibiotic.

Yet another implementation comprises use of a compound of Formula II,wherein R¹ and R² are O⁻M⁺ and each M⁺, independently, is sodium, TRIS,histidine, ethanolamine, diethanolamine, ethylenediamine, diethylamine,triethanolamine, glucamine, N-methylglucamine, ethylenediamine,2-(4-imidazolyl)-ethylamine, choline, or hydrabamine. In a preferredimplementation each M⁺ is sodium.

The method of the invention can further comprise co-administering achemotherapeutic agent, such a cytosine arabinoside (Ara-C), to thesubject. “Co-administration” or “co-administering” can be in the form ofa single formulation (combining, for example, CA4P and a Ara-C withpharmaceutically acceptable excipients, optionally segregating the twoactive ingredients in different excipient mixtures designed toindependently control their respective release rates and durations) orby independent administration of separate formulations containing theactive agents. “Co-administration” further includes concurrentadministration (e.g. administration of CA4P and a Ara-C at the sametime) and time varied administration (administration of CA4P at a timedifferent from that of the Ara-C), as long as both the combretastatinA-4 phosphate, or a pharmaceutically acceptable salt thereof, and thechemotherapeutic agent, e.g., Ara-C, are present in the body intherapeutically effective concentrations during at least partiallyoverlapping times. In preferred implementations the chemotherapeuticagent is Ara-C, etoposide, thioguanine or cyclophosphamide.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and CYTOXAN® (cyclophosphamide);alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol(dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinicacid; a camptothecin (including the synthetic analogue topotecan(HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin,scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); podophyllotoxin; podophyllinic acid; teniposide;cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogues, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gamma1l and calicheamicinomegal1 (see, e.g., Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994));dynemicin, including dynemicin A; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantiobiotic chromophores), aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, caminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMYCIN®,morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin, doxorubicin HCl liposome injection (DOXIL®) anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate,gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), anepothilone, and 5-fluorouracil (5-FU); folic acid analogues such asdenopterin, methotrexate, pteropterin, trimetrexate; purine analogs suchas fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine (Ara-C), dideoxyuridine, doxifluridine, enocitabine,floxuridine; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate;etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine;maytansinoids such as maytansine and ansamitocins; mitoguazone;mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet;pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK®polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane;rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINE®,FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol;pipobroman; gacytosine; arabinoside (“Ara-C”); thiotepa; taxoids, e.g.,paclitaxel (TAXOL®), albumin-engineered nanoparticle formulation ofpaclitaxel (ABRAXANE™), and doxetaxel (TAXOTERE®); chloranbucil;6-thioguanine; mercaptopurine; methotrexate; platinum analogs such ascisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide(VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin;leucovovin; vinorelbine (NAVELBINE®); novantrone; edatrexate;daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoids such as retinoic acid;pharmaceutically acceptable salts, acids or derivatives of any of theabove; as well as combinations of two or more of the above such as CHOP,an abbreviation for a combined therapy of cyclophosphamide, doxorubicin,vincristine, and prednisolone, and FOLFOX, an abbreviation for atreatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU andleucovovin.

As is well-known in the art, solid tumors are quite distinct fromnon-solid tumors, such as those found in hematopoietic-related cancers.Examples of non-solid tumors include leukemias, such as myeloidleukemias and lymphoid leukemias, myelomas, and lymphomas. In someimplementations, the non-solid tumor cell is a hematopoietic neoplasm,which is aberrant growth of cells of the hematopoietic system.Hematopoietic malignancies can have its origins in pluripotent stemcells, multipotent progenitor cells, oligopotent committed progenitorcells, precursor cells, and terminally differentiated cells involved inhematopoiesis. Some hematological malignancies are believed to arisefrom hematopoietic stem cells, which have the ability for self renewal.For instance, cells capable of developing specific subtypes of acutemyeloid leukemia (AML) upon transplantation display the cell surfacemarkers of hematopoietic stem cells, implicating hematopoietic stemcells as the source of leukemic cells. Although hematopoietic neoplasmsoften originate from stem cells, committed progenitor cells or moreterminally differentiated cells of a developmental lineage can also bethe source of some leukemias. For example, forced expression of thefusion protein Bcr/Abl (associated with chronic myelogenous leukemia) incommon myeloid progenitor or granulocyte/macrophage progenitor cellsproduces a leukemic-like condition. In a preferred implementation, thehematopoietic malignancy treated by the method of the invention is acutelymphoblastic leukemia (ALL) or acute myelogenous leukemia (AML).

Hematopoietic neoplasms differ from solid tumors in being capable ofcirculating and having access to various organs through interaction withactivated vascular cells. Indeed, some hematopoietic neoplasms mayadhere to vascular cells, establishing perivascular infiltrates, and assuch may be endowed with a unique mechanism of resistance tochemotherapy. Both circulating and vascular-adherent hematopoieticneoplasms require cytoskeletal stability to maintain mitochondrial andcellular function and avoid cell death. Low and non-toxic doses ofcombretastatin A-4 phosphate can selectively induce apoptosis ofcirculating and vascular-bound leukemic cells, leading to cell death.This induction of apoptosis occurs by a caspase-dependent as well asROS-mediated mitochondrial damage. Thus, combretastatin A-4 phosphate iseffective in treating hematopoietic neoplasms, as demonstrating by itsability to target hematopoietic neoplasms in vitro and in vivo and toeradicate circulating, marrow- and organ-resident vascular-adherenthematopoietic neoplasms.

In some implementations, the hematopoietic neoplasm treated is alymphoid neoplasm, where the abnormal cells are derived from and/ordisplay the characteristic phenotype of cells of the lymphoid lineage.Lymphoid neoplasms can be subdivided into B-cell neoplasms, T andNK-cell neoplasms, and Hodgkin's lymphoma. B-cell neoplasms can befurther subdivided into precursor B-cell neoplasm and mature/peripheralB-cell neoplasm. Exemplary B-cell neoplasms are precursorB-lymphoblastic leukemia/lymphoma (precursor B-cell acute lymphoblasticleukemia) while exemplary mature/peripheral B-cell neoplasms are B-cellchronic lymphocytic leukemia/small lymphocytic lymphoma, B-cellprolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginalzone B-cell lymphoma, hairy cell leukemia, plasma cellmyeloma/plasmacytoma, extranodal marginal zone B-cell lymphoma of MALTtype, nodal marginal zone B-cell lymphoma, follicular lymphoma,mantle-cell lymphoma, diffuse large B-cell lymphoma, mediastinal largeB-cell lymphoma, primary effusion lymphoma, and Burkitt'slymphoma/Burkitt cell leukemia. T-cell and NK-cell neoplasms are furthersubdivided into precursor T-cell neoplasm and mature (peripheral) T-cellneoplasms. Exemplary precursor T-cell neoplasm is precursorT-lymphoblastic lymphoma/leukemia (precursor T-cell acute lymphoblasticleukemia) while exemplary mature (peripheral) T-cell neoplasms areT-cell prolymphocytic leukemia T-cell granular lymphocytic leukemia,aggressive NK-cell leukemia, adult T-cell lymphoma/leukemia (HTLV-1),extranodal NK/T-cell lymphoma, nasal type, enteropathy-type T-celllymphoma, hepatosplenic gamma-delta T-cell lymphoma, subcutaneouspanniculitis-like T-cell lymphoma, Mycosis fungoides/Sezary syndrome,Anaplastic large-cell lymphoma, T/null cell, primary cutaneous type,Peripheral T-cell lymphoma, not otherwise characterized,Angioimmunoblastic T-cell lymphoma, Anaplastic large-cell lymphoma,T/null cell, primary systemic type. The third member of lymphoidneoplasms is Hodgkin's lymphoma, also referred to as Hodgkin's disease.Exemplary diagnosis of this class that can be treated with the compoundsinclude, among others, nodular lymphocyte-predominant Hodgkin'slymphoma, and various classical forms of Hodgkin's disease, exemplarymembers of which are Nodular sclerosis Hodgkin's lymphoma (grades 1 and2), Lymphocyte-rich classical Hodgkin's lymphoma, Mixed cellularityHodgkin's lymphoma, and Lymphocyte depletion Hodgkin's lymphoma. Invarious implementations, any of the lymphoid neoplasms can be treatedwith the combretastatin A-4 phosphate, or a pharmaceutically acceptablesalt thereof.

In some implementations, the hematopoietic neoplasm treated is a myeloidneoplasm. This group comprises a large class of cell proliferativedisorders involving or displaying the characteristic phenotype of thecells of the myeloid lineage. Myeloid neoplasms can be subdivided intomyeloproliferative diseases, myelodysplastic/myeloproliferativediseases, myelodysplastic syndromes, and acute myeloid leukemias.Exemplary myeloproliferative diseases are chronic myelogenous leukemia(e.g., Philadelphia chromosome positive (t(9;22)(qq34;q11)), chronicneutrophilic leukemia, chronic eosinophilic leukemia/hypereosinophilicsyndrome, chronic idiopathic myelofibrosis, polycythemia vera, andessential thrombocythemia. Exemplary myelodysplastic/myeloproliferativediseases are chronic myelomonocytic leukemia, atypical chronicmyelogenous leukemia, and juvenile myelomonocytic leukemia. Exemplarymyelodysplastic syndromes are refractory anemia, with ringedsideroblasts and without ringed sideroblasts, refractory cytopenia(myelodysplastic syndrome) with multilineage dysplasia, refractoryanemia (myelodysplastic syndrome) with excess blasts, 5q-syndrome, andmyelodysplastic syndrome. In various implementations, any of the myeloidneoplasms can be treated with combretastatin A-4 phosphate, or apharmaceutically acceptable salt thereof.

In some implementations, combretastatin A-4 phosphate, or apharmaceutically acceptable salt thereof, can be used to treat acutemyeloid leukemias (AML), which represent a large class of myeloidneoplasms having its own subdivision of disorders. These subdivisionsinclude, among others, AMLs with recurrent cytogenetic translocations,AML with multilineage dysplasia, and other AML not otherwisecategorized. Exemplary AMLs with recurrent cytogenetic translocationsinclude, among others, AML with t(8;21)(q22;q22), AML1 (CBF-alpha)/ETO,Acute promyelocytic leukemia (AML with t(15;17)(q22;q11-12) andvariants, PML/RAR-alpha), AML with abnormal bone marrow eosinophils(inv(16)(p13q22) or t(16;16)(p13;q11), CBFb/MYH11X), and AML with 11q23(MLL) abnormalities. Exemplary AML with multilineage dysplasia are thosethat are associated with or without prior myelodysplastic syndrome.Other acute myeloid leukemias not classified within any definable groupinclude, AML minimally differentiated, AML without maturation, AML withmaturation, acute myelomonocytic leukemia, acute monocytic leukemia,acute erythroid leukemia, acute megakaryocytic leukemia, acutebasophilic leukemia, and acute panmyelosis with myelofibrosis.

One aspect of the invention is a pharmaceutical composition useful fortreating a hematopoietic neoplasm in a warm-blooded animal, whichcomposition comprises combretastatin A-4 phosphate, or apharmaceutically acceptable salt thereof, in combination with apharmaceutically acceptable excipient. The composition is prepared inaccordance with known formulation techniques to provide a compositionsuitable for oral, topical, transdermal, rectal, by inhalation,parenteral (intravenous, intramuscular, or intraperitoneal)administration, and the like. Detailed guidance for preparingcompositions of the invention can be found by reference to the 18^(th)or 19^(th) Edition of Remington's Pharmaceutical Sciences, Published bythe Mack Publishing Co., Easton, Pa. 18040. In certain implementations,the pharmaceutical composition further comprises a chemotherapeuticagent, such as Ara-C, etoposide, thioguanine or cyclophosphamide.

Unit doses or multiple dose forms are contemplated, each offeringadvantages in certain clinical settings. The unit dose would contain apredetermined quantity of active compound calculated to produce thedesired effect(s) in the setting of treating cancer. The multiple doseform may be particularly useful when multiples of single doses, orfractional doses, are required to achieve the desired ends. Either ofthese dosing forms may have specifications that are dictated by ordirectly dependent upon the unique characteristic of the particularcompound, the particular therapeutic effect to be achieved, and anylimitations inherent in the art of preparing the particular compound fortreatment of cancer.

A unit dose will contain a therapeutically effective amount sufficientto treat a hematopoietic neoplasm in a subject and may contain fromabout 1.0 to 1000 mg of compound, for example about 50 to 500 mg.

The combretastatin A-4 phosphate, or a pharmaceutically acceptable saltthereof, preferably is administered parenterally, e.g., intravenously,intramuscularly, intravenously, subcutaneously, or intraperitoneally.The carrier or excipient or excipient mixture can be a solvent or adispersive medium containing, for example, various polar or non-polarsolvents, suitable mixtures thereof, or oils. As used herein “carrier”or “excipient” means a pharmaceutically acceptable carrier or excipientand includes any and all solvents, dispersive agents or media,coating(s), antimicrobial agents, iso/hypo/hypertonic agents,absorption-modifying agents, and the like. The use of such substancesand the agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active ingredient, use in therapeutic compositionsis contemplated. Moreover, other or supplementary active ingredients canalso be incorporated into the final composition.

Solutions of the compound may be prepared in suitable diluents such aswater, ethanol, glycerol, liquid polyethylene glycol(s), various oils,and/or mixtures thereof, and others known to those skilled in the art.

The pharmaceutical forms suitable for injectable use include sterilesolutions, dispersions, emulsions, and sterile powders. The final formmust be stable under conditions of manufacture and storage. Furthermore,the final pharmaceutical form must be protected against contaminationand must, therefore, be able to inhibit the growth of microorganismssuch as bacteria or fungi. A single intravenous or intraperitoneal dosecan be administered. Alternatively, a slow long term infusion ormultiple short term daily infusions may be utilized, typically lastingfrom 1 to 8 days. Alternate day or dosing once every several days mayalso be utilized.

Sterile, injectable solutions are prepared by incorporating a compoundin the required amount into one or more appropriate solvents to whichother ingredients, listed above or known to those skilled in the art,may be added as required. Sterile injectable solutions are prepared byincorporating the compound in the required amount in the appropriatesolvent with various other ingredients as required. Sterilizingprocedures, such as filtration, then follow. Typically, dispersions aremade by incorporating the compound into a sterile vehicle which alsocontains the dispersion medium and the required other ingredients asindicated above. In the case of a sterile powder, the preferred methodsinclude vacuum drying or freeze drying to which any required ingredientsare added.

In all cases the final form, as noted, must be sterile and must also beable to pass readily through an injection device such as a hollowneedle. The proper viscosity may be achieved and maintained by theproper choice of solvents or excipients. Moreover, the use of molecularor particulate coatings such as lecithin, the proper selection ofparticle size in dispersions, or the use of materials with surfactantproperties may be utilized.

Prevention or inhibition of growth of microorganisms may be achievedthrough the addition of one or more antimicrobial agents such aschlorobutanol, ascorbic acid, parabens, thermerosal, or the like. It mayalso be preferable to include agents that alter the tonicity such assugars or salts.

Another aspect of this invention is a method for treating ahematopoietic neoplasm in a warm-blooded animal, which method comprisesadministering a therapeutically effective amount of combretastatin A-4phosphate, or a pharmaceutically acceptable salt thereof. Thecombretastatin A-4 phosphate, or a pharmaceutically acceptable saltthereof, can be administered to an appropriate subject in atherapeutically effective dose by a medically acceptable route ofadministration such as orally, parentally (e.g., intramuscularly,intravenously, subcutaneously, interperitoneally), transdermally,rectally, by inhalation and the like.

With mammals, including humans, the effective amounts can beadministered on the basis of body surface area. The interrelationship ofdosages varies for animals of various sizes and species, and for humans(based on mg/m² of body surface) is described by E. J. Freireich et al.,Cancer Chemother. Rep., 50(4):219 (1966). Body surface area may beapproximately determined from the height and weight of an individual(see, e.g., Scientific Tables, Geigy Pharmaceuticals, Ardsley, N.Y. pp.537-538 (1970)). A suitable dose range is from 1 to 1000 mg ofequivalent per m² body surface area of a compound of the invention, forinstance from 50 to 500 mg/m².

Another important feature of the method provided by the presentinvention relates to the relatively low apparent overall toxicity of thederivatives administered in accordance with the teachings herein.Overall toxicity can be judged using various criteria. For example, lossof body weight in a subject over 10% of the initially recorded bodyweight (i.e., before treatment) can be considered as one sign oftoxicity. In addition, loss of overall mobility and activity and signsof diarrhea or cystitis in a subject can also be interpreted as evidenceof toxicity.

VI. EXAMPLES A. Example 1 Combretastatin A4 Phosphate (CA4P) InhibitsLeukemic Cell Proliferation

Leukemic cells were seeded at 1×10⁵ cells/ml in X-vivo medium(Bio-Whittaker, MA) with 5% FBS and CA4P or pre-incubated with thePARP-inhibitor DPQ or caspase-inhibitors Z-VAD-fmk and Q-VD-OPh (R&D,Minneapolis, Minn.). A panel of leukemic cells was incubated with CA4Pat the different concentrations indicated in FIG. 1, and viable cellswere counted after 48 hours using trypan blue exclusion. Results of fourexperiments in duplicate are expressed as the ratio of the percentage ofviable cells/control±SEM (*p<0.05 as compared with CA4P-untreated cells;n=4). After incubation for 48 hours, cells were counted usingtrypan-blue exclusion. CA4P at concentrations as low as 1 nM initiatedcell death of non-adherent, anchorage-independent AML cells in vitro,with the IC₅₀ ranging from 2.5 to 5 nM (FIG. 1). The majority of the AMLcell lines tested was sensitive to CA4P at a concentration of 2.5 nM orless. All leukemic cell lines, as well as a recently established primaryleukemic cell line (R81) 16 were sensitive at low doses of CA4P (<10nM).

B. Example 2 CA4P Causes Cell Cycle Arrest in G2/M Phase

CA4P induces G2/M arrest and cell death, as evidenced by increase in thesub-G0/G1 peak (FIG. 2). Leukemic cells were seeded at 10⁵ per ml inX-vivo supplemented with 5% FBS and then incubated with CA4P. KG1aleukemic cells were treated with CA4P at 0, 5 and 10 nM concentrations.After incubation for 48 hours, apoptotic cells were quantified byApoAlert Annexin-V-fluorescine isothiocyanate (FITC) propidium iodide(PI) Apoptosis Kit (BD) using a Coulter Elite flow cytometer. InCA4P-treated cells, cell cycle analysis with propidium iodine (PI)showed G₂/M arrest and evidence of DNA fragmentation (sub G₀-phase) at48 hours.

C. Example 3 Combretastatin A-4 Phosphate Causes DNA Fragmentation andMorphological Evidence of Mitotic Catastrophe

DNA damage in CA4P-incubated leukemic cells was assessed by comet assay.The concept behind this assay is based upon the ability of denatured,cleaved DNA fragments to migrate out of the cell under the influence ofan electric field, whereas undamaged DNA migrates more slowly andremains within the confines of the nucleus. Evaluation of the DNA“comet” tail shape and migration pattern allows for assessment of DNAdamage. Results were expressed as the percentage of cells with a comettail in 100 randomly selected, non-overlapping cells visualized bystandard light microscopy. Quantification of the number of leukemiccells displaying a comet tail strongly increased after CA4P treatment(FIG. 3), consistent with CA4P-induced DNA damage. Results of threeexperiments in duplicate are expressed as the mean of the number ofcells with a comet tail (%)±SEM (*p<0.05 as compared with CA4P-untreatedcells; n=3).

D. Example 4 CA4P Induces Cell Death Through Apoptosis without Evidenceof Necrosis

CA4P-treated AML cells were subjected to Annexin-V/propidium iodide (PI)staining and quantification by flow cytometry. Combretastatin A-4phosphate (CA4P) induces caspase-independent apoptosis in leukemiccells. CA4P induces apoptosis of leukemic cells. Leukemic cells weretreated with or without CA4P for 48 hours and the percentage ofapoptotic leukemic cells was determined by Annexin V and propidiumiodide staining using flow cytometry. Results are representative ofthree independent experiments. In contrast to endothelial cells, CA4Pinduced phosphatidylserine externalization in all leukemic cell linestested, suggesting that CA4P promotes leukemic cell death throughapoptosis (FIG. 4). In the majority of AML cell lines tested, CA4P at aconcentration of 5 nM induced phosphatidylserine externalization(Annexin V+) in more than 50% of the leukemic cells. Only a small numberof PI+, Annexin V(−) cells were detected, suggesting that CA4P-mediatedcell death is apoptotic rather than necrotic.

Nonetheless, to rule out the possibility that CA4P may trigger necroticcell death in subsets of the leukemic cells, the release of nuclear HMG1protein into the culture supernatant, which has been described as asensitive and highly specific marker for necrotic cell death (Scaffidi,et al. 2002. Nature 418, 191-5), was quantified. For detection of HMG1protein, KG1a leukemic cells (1×106 in 2 ml X-vivo/5% FBS) wereincubated with 100 μM staurosporine to induce apoptosis, freeze-thawedthree times to induce necrosis, or treated with 20 mM CA4P for 24 hours.Cells were spun down, pelleted and the supernatant subjected to Westernblot for HMG135. Western blot shows release of pelleted (P) nuclear HMG1protein into the supernatant (S) in necrotic (freeze-thaw), but notapoptotic (staurosporine-treated) or CA4P-treated KG1a leukemic cells(data not shown). Identical results were obtained in three independentexperiments.

E. Example 5 CA4P-Induced Cell Death is Partially Caspase-Dependent

Both Z-VAD-fmk and Q-VD, two potent general caspase inhibitors, blockedCA4P-induced caspase-3 activation, but only partially affectedCA4P-induced hematopoietic neoplasm death, (FIG. 5). Leukemic cells weretreated with or without CA4P for 48 hours in presence of caspaseinhibitor Q-VD and the percentage of apoptotic leukemic cells wasdetermined by Annexin V and propidium iodide staining using flowcytometry. Percentage of alive Annexin V(−)/PI(−) cells was plotted.Results are average of three independent experiments (*p=0.008 ascompared with CA4P-treated cells). Quantification of apoptosis byAnnexin V/PI staining showed that only 33% of the CA4P induced apoptosiswas blocked by Q-VD in HL60 cells (FIG. 5). Z-VAD-fmk had no effect(data not shown). These data suggest that CA4P induces apoptosis ofhematopoietic neoplasms through caspase-dependent andnon-caspase-dependent pathways.

F. Example 6 CA4P Decreases Mitochondrial Transmembrane Potential

To determine mitochondrial membrane potential, leukemic cells incubatedwith CA4P for 48 hours were harvested, and incubated for 10 minutes at37° C. in serum-free culture medium at a concentration of 2×105 cells/mlwith 20 nM of 3,30-dihexyloxacarbocyanine (DiOC6(3), Molecular Probes),a cell-permeant, green-fluorescent, lipophilic dye that is selective forthe mitochondria of viable cells. Cells were collected by centrifugationand analyzed by flow cytometry.

The pattern of DiOC₆(3) fluorescence taken up by control leukemic cellsshowed cell populations with bright fluorescence, representing cellswith intact high MTP (FIG. 6). Results are representative of threeindependent experiments. In contrast, the amount of DiOC₆(3) dye takenup in CA4P-treated leukemic cells was strongly decreased. The percentageof cells with fluorescence below control ranged from 12 to 89% of totalcells, and for each cell line tested, the results correlated well withthe extent of Annexin-V positivity. These data indicated thatCA4P-induced leukemic cell death is possibly mediated through alterationof mitochondrial permeability.

Mitochondrial damage may result in the release of pro-apoptoticmitochondrial membrane proteins (MMPs) such as cytochrome c, SMAC/diabloand ARTS. By immunofluorescence, we observed the release of cytochrome cand ARTS from mitochondria in leukemic cells after CA4P exposure (datanot shown), consistent with mitochondrial destabilization. Cytochrome cand ARTS were detected using mAb clone 6H2.B4 (1:100, BD Pharmingen) andpolyclonal-antibody A3720 (1:50, Sigma), both followed by AlexaFluor 488conjugated secondary antibody (1:200, Molecular Probes) and analyzed byconfocal microscopy.

G. Example 7 Reactive Oxygen Species (ROS) Accumulation

Leukemic cells were treated with 5 nM (HL60, R81, U937) or 10 nM (KG1a,THP-1) for 48 hours with the ascorbic acid concentrations indicated andviable cells determined by trypan blue exclusion. Results of threeexperiments in triplicate are expressed as percentage of viable cellscompared to control±SEM (*p<0.05 as compared with CA4P-untreated cells;n=3). Co-incubation of leukemic cells with the antioxidant ROSscavenger, ascorbic acid (AA), led to a partial decrease inCA4P-mediated cell death (FIG. 7).

In addition, rapid accumulation of superoxide anions after CA4Pexposure, as evidenced by an increase in intracellular fluorescenceafter H₂DCFDA loading and reversal of ROS accumulation by ascorbic acid(FIG. 8), was also detected. Intracellular ROS were detected aspreviously described (Krejsa & Schieven, Methods Mol Biol 99:35-47(2000)). Briefly, leukemic cells were loaded with 2 μM H2DCFDA(Molecular Probes) in assay buffer (RPMI with 10 mM HEPES) for 30 min.at 37° C. and mean fluorescent intensity was measured by flow cytometry.Results are shown from triplicate measurements (±SEM) and arerepresentative of three independent experiments (*p<0.05 as comparedwith untreated cells; n=3).

HL-60 cells were treated with or without CA4P for 48 hours in presenceof another ROS scavenger, deferoxamine (DFX), and caspase inhibitor Q-VDand the percentage of apoptotic leukemic cells was determined by AnnexinV and propidium iodide staining using flow cytometry. Results areaverage of three independent experiments (*p=0.01 as compared withCA4P-treated cells, *p=0.04 as compared to CA4P+Q-VD treated cells). DFXalso slightly inhibited CA4P-induced cell death and had an additiveeffect with caspase inhibitor Q-VD in HL60 cells, inhibiting up to 71%of CA4P-induced cell death (FIG. 9). Taken together, these data indicatethat CA4P induces cell death in part through a caspase-dependent as wellas in part through a non-caspase dependent cell pathway, by accumulationof ROS as a result of tubulin-destabilization and disruption of themitochondrial respiratory chain.

H. Example 8 Subcutaneous In Vivo Leukemia Model

HL60 (5×10⁶ cells) were injected subcutaneously into the dorsa ofseven-week-old NOD-SCID mice (Jackson Laboratory). When mice bore atumor (i.e. after 12 days), 4 experimental groups were randomized, eachwith 9 animals. Daily treatment was initiated at this time: the CA4Pgroups were subjected to intraperitoneal injection of CA4P at 10, 25 and50 mg/kg body weight, and the control group received PBS. After a 3-daytreatment, animals were sacrificed, tumors removed and then subjected toimmunohistochemical analysis. Tumors were embedded in paraffin, seriallysectioned, and stained with hematoxylin and eosin for histologicalanalysis. Cell death was assessed by TUNEL assay. Cell death withinparaffin-embedded tumor sections was detected by TUNEL reaction (RocheDiagnostics). The detection of cell death in this assay relies on thedetection of free 3′OH DNA ends. Positive signal was revealed by fastred and tumor sections analyzed by light microscopy after hematoxylincounterstain.

Tumors from mice treated with CA4P for 3 days were softer andhemorrhagic (particularly when treated with 50 mg/kg) than thoseobserved in the control group. Tumor sections of the control untreatedmice showed large areas of viable HL60 cells without significantnecrosis or fibrosis. In contrast, all tumors in CA4P treated mice werelargely necrotic. The control tumor sections were negative for TUNELreaction. In sharp contrast, most of the leukemic cells were non-viablefollowing treatment with low to high doses of CA4P (data not shown). Theextent of intra-tumor vascularization within the different groups wasassessed by immunostaining for the endothelial-specific antigen MECA-32.Control tumors exhibited abundant number of vessels, whereasCA4P-treated tumors showed only a very small number of neo-vessels (FIG.10, p<0.05, n=5). The neo-vessel density was evaluated by microscopiccounting of 5 fields at 10× magnification, and presented as mean numberof microvessels per microscopic field±SEM (*p<0.05 as compared with thePBS control group; n=5). The decrease in intra-tumor vascularizationinduced by CA4P was dose dependent (54±13%, 68±6% and 90±3% decrease for10, 25 and 50 mg/kg, respectively). These data suggest that targetingleukemic xenografts is associated with tumor regression and inhibitionof neo-angiogenesis.

I. Example 9 Systemic In Vivo Leukemia Model

NOD-SCID mice were intravenously inoculated with 1×10⁷ GFP+HL60 cells.The HL60 cells were labeled with green fluorescent protein (GFP) by alentiviral construct, and inoculated systemically through tail veininjection. Three days after inoculation, mice were divided into 2 groupsof five mice. One group was treated every other day with PBS (control)and the second group received 25 mg/kg CA4P every other day. Eachexperiment was done three times. At day 30 after the start of theexperiment, two mice from each group were killed, and their organs(spleen, liver and lung), peripheral blood and marrow of surgicallyremoved femurs were collected, and analyzed for the presence of humanleukemic cells by flow cytometry. Single cell suspension was stainedusing phytoerythrin-labeled anti-human CD45 mAb (Pharmingen), and thepercentage of double positive human CD45-PE and GFP cells was determinedflow cytometer. The extent of GFP+HL60 cell infiltration was assessed byfluorescence microscopy. In a second set of experiments, GFP+U937 cellswere used instead of HL60 cells, and the animals sacrificed after 30days.

In the untreated control group, the mice survived for 32-40 days onlyand succumbed to systemic spread of leukemia. In contrast, CA4Ptreatment significantly increased survival of the intravenouslyxenotransplanted mice (FIG. 11, p<0.05; n=5). This increase in survivalwas accompanied by a decrease in circulating leukemic cells observed 30days after leukemic cell injection, as assessed by flow cytometrydetermination of double positive cells for human CD45 and GFP (0.9% forcontrol vs. 0.2% for CA4P treatment, p<0.05, n=5) (FIG. 12). Thirty daysafter xenotransplantation of GFP+HL60 cell injection, the presence ofGFP+HL60 cells in the peripheral blood and the different tissues of themice was assessed by quantification of double positive GFP and humanCD45 cells by flow cytometry. Remarkably, engraftment of leukemic cellsin the bone marrow was completely eradicated in the CA4P-treated mice(FIG. 12). In addition, there was no evidence of infiltration ofGFP+HL60 cells in the spleen and the lung of CA4P-treated mice, whereasa small population (0.1%) of human CD45 and GFP positive cells wasdetected in the liver (FIG. 12). In contrast, control mice had massiveleukemic infiltrates in the spleen (5.2%), liver (2.6%) and lung (3.5%)(FIG. 12). Concurrent histological analysis showed the presence ofleukemic infiltrates in spleen, liver and lung sections of control mice,but only minimal foci of leukemic cells in the liver of CA4P-treatedmice, confirming a drastic decrease in the amount of residual disease.

To confirm these results with a different leukemic cell line, theexperiments were repeated using GFP-labeled U937 cells and harvestedorgans after 30 days. The results obtained were similar to theexperiment using HL60 cells: in the treatment group, less than 0.05%GFP-positive U937 leukemic cells were detectable in the liver and bonemarrow, whereas the control animals had significant leukemic organinfiltration present in the spleen, liver and lungs (data not shown).These data show that CA4P can efficiently block systemic hematopoieticneoplasm growth in vivo and inhibit organ-specific spread ofhematopoietic neoplasms, apparently through disruption of hematopoieticneoplasm growth, migration and possibly interfering with the activationof vascular stromal cells.

J. Example 10 CA4P Modulates Cell Adhesion

CA4P may modulate leukemia-vascular interactions, thereby disruptingchemo-protected niches for leukemic cells. Indeed, CA4P treatment ofleukemia xenografts significantly reduced expression of VCAM-1, a VLA4ligand and key molecule in leukemia-stroma adhesion. NOD-SCID mice withsubcutaneous HL60 AML tumors were treated with CA4P or PBS (untreatedcontrol). Immunofluorescence for VCAM-1 showed significantly decreasedVCAM-1 expression in CA4P-treated tumors (data not shown).

Human umbilical vein endothelial cells (HUVECs) were activated withIL-1β (5 ng/ml) for 24 hours with CA4P added at concentrations from 0 to5 nM. VCAM-1 expression was determined by flow cytometry withphytoerythrin-conjugated anti-CD106 (VCAM-1) mAb. Treatment of HUVECswith low, non-toxic (1 to 5 nM range) doses of CA4P significantlyreduced expression of VCAM-1 (FIG. 13), without inducing apoptosis.

To assess leukemic cell adhesion, 1×10⁵ GFP+HL60 or U937 cells inX-vivo/5% FBS were added per well. The percentage of GFP++adherent cellswas quantified by fluorescent microscopy. To compare the resistance ofvascular-adherent to non-adherent leukemic cells in co-culture, leukemiccells were seeded on either IL-1β-activated or non-activated HUVECs,with addition of CA4P. After 48 hours, GFP++leukemic cells were removedfrom the wells by trypsinization and quantified by fluorescencemicroscopy and flow cytometry. Treatment with CA4P led to a decreasedattachment of HL60 and U937 AML cells to HUVECs in vitro (FIGS. 14A and14B). The number of adherent cells is expressed as a percentage of totalleukemic cells and representative of three independent experimentsperformed in triplicate SEM (*p<0.05 as compared to CA4P-untreatedcontrol).

In turn, decreased adhesion to HUVECs rendered the leukemic cells moresensitive to subsequent CA4P treatment (FIGS. 15A and 15B). Survival ofGFP+U937 (E) and HL60 (F) leukemic cells co-cultured with HUVECs isexpressed as a percentage of total cells. Adherent (i.e. attached toIL-1β activated HUVECs) and non-adherent leukemic cells (i.e. culturedwith non-activated HUVECs) are depicted. Results are representative ofthree independent experiments performed in triplicate±SEM (*p<0.05).Taken together, these findings suggest that CA4P reduces expression ofVCAM-1 on vascular cells, thereby increasing the chemosensitivity ofhematopoietic neoplasms. Most notably, low CA4P concentrations reducedthe expression of VCAM-1 without inducing endothelial cell death,suggesting that CA4P exerts an anti-leukemic effect by modulatingadhesive function of the endothelial cells prior to its anti-angiogeniceffect.

K. Example 11 CA4P has Minimal Bone Marrow Toxicity

Eight week-old sex matched CD1 mice (Jackson Laboratory, Bar Harbor,Me.) were treated with CA4P at 25 mg/kg subcutaneously every other dayfor 4 weeks. Serial complete blood counts were monitored using BayerADVIA 120 hematology analyzer. the mice so treated for 4 weeks displayeda slight decrease in WBC and absolute neutrophil count only, suggestingminimal marrow suppression.

In addition, human umbilical cord derived (CB) CD34+ cells cultured invitro in the presence of kit-ligand (Stem Cell Factor) and CA4P for 48 hproduced only minimal effect on cell viability as assayed by Annexin/PIstaining. Cord blood CD34+ cells were isolated by magnetic procedure andcultured with recombinant human SCF (50 ng/ml, Peprotech) and CA4P for48 h. Annexin/PI staining was then performed. CD34+ cells were alsocultured in methylcellulose supplemented with cytokines and CA4P (Stemcell technologies, Vancouver, Canada) for 14 days. Colonies were scored.Moreover, CA4P did not impair colony-forming potential of CD34+ cells,demonstrating that CA4P at the concentrations used to targethematopoietic neoplasms has no major toxic effect on normal stem orprogenitor cell function. As such, CA4P used as single agent canselectively target circulating and or tissue-resident hematopoieticneoplasms without incurring significant hematological toxicity.

1. A method of treating a hematopoietic neoplasm comprisingadministering a therapeutically effective amount of combretastatin A-4phosphate (CA4P), or a pharmaceutically acceptable salt thereof, to asubject having a hematological malignancy.
 2. The method of claim 1,wherein the combretastatin A-4 phosphate salt is a compound of theFormula II:

wherein each OR¹ and OR² independently is selected from OH, —O⁻QH⁺ and—O⁻M⁺, wherein M⁺ is a monovalent or divalent metal cation, and Q is: a)an amino acid containing at least two nitrogen atoms where one of thenitrogen atoms, together with a proton, forms a quaternary ammoniumcation QH⁺; or b) an organic amine containing at least one nitrogen atomwhich, together with a proton, forms a quaternary ammonium cation, QH⁺.3. The method of claim 2, wherein one of OR¹ and OR² is hydroxyl and theother is —O⁻QH⁺ and Q is tromethamine or L-histidine.
 4. The method ofclaim 1, wherein the combretastatin A-4 phosphate salt is a compound ofthe Formula II:

wherein each OR¹ and OR² independently is selected from OH, and —O⁻M⁺,wherein each M⁺ independently is an aliphatic organic amine, an alkalimetal, a transition metal, a heteroarylene, a heterocyclyl, anucleoside, a nucleotide, an alkaloid, an amino sugar, an amino nitrile,or an nitrogenous antibiotic.
 5. The method of claim 4, wherein each M⁺,independently, is selected from the group consisting of sodium, TRIS,histidine, ethanolamine, diethanolamine, ethylenediamine, diethylamine,triethanolamine, glucamine, N-methylglucamine, ethylenediamine,2-(4-imidazolyl)-ethylamine, choline, and hydrabamine.
 6. The method ofclaim 1, wherein the hematopoietic neoplasm is a myeloid neoplasm. 7.The method of claim 6, wherein the hematopoietic neoplasm is an acutemyeloid leukemia (AML).
 8. The method of claim 1, wherein thehematopoietic neoplasm is a lymphoid neoplasm.
 9. The method of claim 1,wherein the hematopoietic neoplasm is a refractory organ-infiltratingleukemia.
 10. The method of claim 1, wherein the pharmaceuticallyacceptable salt is a tromethamine salt of combretastatin A-4 phosphate.11. The method of claim 1, further comprising co-administering achemotherapeutic agent to the subject having a hematological malignancy.12. The method of claim 11, wherein the chemotherapeutic agent is Ara-C,etoposide, thioguanine or cyclophosphamide.